Cloning and characterization of the nuclear gene and cDNAs for triosephosphate isomerase of the marine red alga Gracilaria verrucosa

cDNAs and an intronless single-copy nuclear gene (TPI1) encoding triosephosphate isomerase have been cloned and sequenced from the marine red alga Gracilaria verrucosa. The predicted amino-acid sequence of TPI1 is readily alignable with those of other known TPIs; 26 of 27 active-site residues and 19 of 26 intersubunit-contact residues are identical between TPIs of G. verrucosa and/or animals and green plants. A partial cDNA sequence of a second TPI gene (TPI2), presumably encoding plastid-localized TPI, was recovered by PCR and demonstrated by phylogenetic analysis to be red algal; no TPI2 cDNA or genomic clones could be recovered. Genomic Southern analysis demonstrated that at least two TPI-like genes are present in the nuclear DNA of G. verrucosa.Issued as NRCC no. 38070 GSDB accession L38662     

Identification and localization of two genes on the chicken Z chromosome: implication of evolutionary conservation of the Z chromosome among avian species

A cDNA clone containing an insert of about 3.4 kb, pCIREBP, was isolated from the chicken liver cDNA library and identified as a clone for the chicken homologue of iron-responsive element-binding protein (IREBP). The deduced amino acid sequence showed 88% identity with that of the mouse IREBP and 17 out of the 20 active site residues of the pig heart mitochondrial aconitase were conserved. Another cDNA clone, pZOV3, containing an insert of about 4.5 kb was isolated from the chicken ovary cDNA library. This cDNA contained an open reading frame for 327 amino acid residues, whose sequence had partial similarity to two immunoglobulin superfamily proteins; mouse GP-70 and chicken HT7. Fluorescencein situ hybridization using corresponding genomic clones revealed that both genes are localized on the Z chromosome; the ZOV3 gene at the middle of the short arm and the IREBP gene at the boundary of heterochromatin on the long arm. Southern blot hybridization to male and female genomic DNA preparations from six species representing five avian genera suggested that these two genes are Z-linked in all the species tested.     

Cloning and sequence analysis of the glucoamylase gene of Neurospora crassa

A 1.0-kb DNA fragment, corresponding to an internal region of the Neurospora crassa glucoamylase gene, gla-1, was generated from genomic DNA by the polymerase chain reaction, using oligonucleotide primers which had been deduced from the known N-terminal amino-acid sequence or from consensus regions within the aligned amino-acid sequences of other fungal glucoamylases. The fragment was used to screen an N. crassa genomic DNA library. One clone contained the gene together with flanking regions and its sequence was determined. The gene was found to code for a preproprotein of 626 amino acids, 35 of which constitute a signal and propeptide region. The protein and the gene are compared with corresponding sequences in other fungi.     

Isolation and characterization of the cerato-ulmin toxin gene of the Dutch elm disease pathogen,Ophiostoma ulmi

The hydrophobic protein cerato-ulmin (CU), produced by Ophiostoma ulmi, has been implicated in the pathogencity of this fungus on elm. Primers were designed based on the nucleotide sequence deduced from the published CU amino-acid sequence, and a DNA fragment of the cu gene was amplified using the polymerase chain reaction. The amplified cu fragment was used as a hybridization probe to identify and isolate the cu gene from a genomic DNA library of an aggressive isolate of O. ulmi (= O. novo-ulmi). The cu coding region is interrupted by two introns and encodes a 100 amino-acid prepro-CU polypeptide that is processed to a 75 amino-acid mature protein upon secretion. CU shows significant sequence similarity to hydrophobins secreted by certain other fungi.This paper is dedicated to our late friend and colleague, Shozo TakaiCommunicated by O. C. Yoder     

Use of the polymerase chain reaction to identify coding sequences for chitin synthase isozymes in Phialophora verrucosa

Based on conserved amino-acid regions predicted for the chitin synthases (Chs) of Saccharomyces cerevisiae, two different primer sets were synthesized and used in polymerase chain reactions (PCRs) to amplify 614-bp and 366-bp sequences from genomic DNA of the zoopathogenic fungus Phialophora verrucosa. DNA-sequencing and Southern-blotting analyses of the 614-bp DNA amplification products suggested that portions of two distinct P. verrucosa chitin synthase genes (PvCHS1, PvCHS2), coding for two different zymogenic-type PvChs isozymes, had been identified. The deduced amino-acid sequence of each fell into different Chs classes, namely class I and class II. In addition, the 366-bp DNA segment was shown to code for a conserved region having homology with the CSD2/CAL1 gene of S. cerevisiae, which encodes a nonzymogenic-type enzyme, Chs3, in that fungus. The amino-acid sequence derived from PvCHS3 exhibits 88.2% similarity and 78.4% identity to the same amino-acid region of the S. cerevisiae enzyme. These results provide a critical first step toward investigating the molecular and pathogenic importance of CHS gene regulation in this fungus and for exploring steps leading to Chs function as potential targets for the design of new therapeutic agents.     

Cloning and nucleotide sequence of the genomic ribonuclease T2 gene (rntB) from Aspergillus oryzae

Summary Using synthetic oligonucleotide probes, we have cloned a genomic DNA sequence encoding a ribonuclease (RNase) T₂ gene (rntB) from Aspergillus oryzae on a 4.8 kb HindIII fragment. DNA sequence analysis of the RNase T₂ revealed the following: (1) The gene is arranged as five exons and four introns; (2) The deduced amino acid sequence contains 239 amino acid residues of the mature enzyme. In addition, there exist 17 amino acid residues thought to be a signal peptide sequence at the N-terminus and 20 amino acid residues at the C-terminus; (3) The nucleotide sequence of the rntB gene is homologous to those of the RNase Rh gene from Rhizopus niveus and the S2 stylar glycoprotein gene of Nicotiana alata with degree of about 51% and 47%, respectively; (4) A. oryzae and A. nidulans transformed with the cloned rntB gene had much higher ribonuclease T₂ activity than wild-type strains.     

Cloning and expression of a chitinase gene from the hyperparasitic fungus Aphanocladium album

Summary Recombinant clones from a cDNA library of an Aphanocladium album chitinase-overproducing mutant strain were isolated by screening with antiserum against a 39 kDa chitinase purified from this hyperparasitic fungus. Analysis of the isolated positive clones indicated that most of them carried the same cDNA. A cDNA from this group was used as a hybridization probe to isolate an 8 kb DNA fragment from a genomic library of the wild-type strain. The chitinase 1 gene was mapped to this fragment by two independent approaches. Its partial DNA sequence was in perfect agreement with an amino-terminal peptide sequence obtained by sequencing 23 amino acids of the 39 kDa chitinase. Its transfer in Fusarium oxysporum resulted in a transformant producting both a protein of about 39 kDa that cross-reacted with the chitinase antiserum and a chitinase activity that was inhibited by the same antiserum. Northern blot analysis indicates that the cloned chitinase gene was subject to catabolite repression and appeared inducible by chitin.     

Genomic organization and mapping of the human activin receptor type IIB (hActR-IIB) gene

Activins, members of a family of proteins that includes transforming growth factor-beta (TGF-beta), are gonadal polypeptide hormones that stimulate secretion of follicle-stimulating hormone (FSH). During large-scale sequencing analysis of a 1.2-Mb fragment of human genomic DNA on 3p22–p21.3, we found the gene encoding activin receptor type IIB (hActR-IIB). Comparison of its reported cDNA sequence with this genomic sequence showed that the hActR-IIB gene consists of 11 exons and spans about 30 kb of genomic DNA. Received: August 11, 1997/Accepted: October 22, 1997     

Analysis of sequence polymorphism of a major mite allergen, Der p 2

Background The major house dust mite allergen Der p 2 has been regarded as an important allergen involved in the immunopathogenesis of allergic asthma and eczema. Objectives To determine the degree of sequence polymorphism exists in Der p 2 at both the genomic DNA and cDNA levels. Methods Isolation of cDNA clones was performed by screening the cDNA libraries with Der p 2 cDNA probe and the genomic sequences for Der p 2 were obtained by PCR amplification from environmental dust mites with Der p 2-specific primers. DNA sequencing was carried out by the dideoxynucleotide chain termination method. The study of Der p 2 isoforms was performed by 2-D gel immunoblot analysis using mouse anti-Der p 2 serum. Results In this study, we have characterized the seqtiences of three difierent genealleles coding for the major house dust mite allergen Der p 2 at the cDNA level. The translated polypeptides from these clones differed from each other by 3–4 amino-acid residues. These polymorphic residues determined were also found in Der f 2 and they were located in regions containing T-epitopes. In addition, the genomic DNA sequences of Der p 2 which were obtained by PCR amplification using the environmental mites isolated from Taiwan and Australia have helped to confirm the authenticity of the polymorphisms detected in the cDNA clones generated from CSL cultured mites. Furthermore, 2D- immunoblot analysis indicated that there were at least 10 different isoforms (p 1 values range from greater than 7.0–5.9) of Der p 2 proteins produced by CSL cultured mites. Conclusion The results showed that there was a small but significant degree of sequence polymorphisms exists in Der p 2 gene alleles. Interestingly, the polymorphic residues were found in regions containing previously determined T-epitopes. The polymorphism data reported here will be important for the understanding of the immune responses of mite allergens as well as for the development of the peptide-based immunotherapeutic reagents for mite allergy.     

Identification and characterization of PaMTH1, a putative O-methyltransferase accumulating during senescence of Podospora anserina cultures

A differential protein display screen resulted in the identification of a 27-kDa protein which strongly accumulates during the senescence of Podospora anserina cultures grown under standard conditions. After partial determination of the amino-acid sequence by mass-spectrometry analysis of trypsin-generated fragments, pairs of degenerated primers were deduced and used to amplify parts of the sequence coding for the protein. These PCR products were utilized to select specific cDNA and genomic clones from DNA libraries of P. anserina. A subsequent DNA-sequence analysis revealed that the 27-kDa protein is encoded by a discontinuous gene, PaMth1, capable of coding for 240 amino acids. The first three amino-terminal residues appear to be removed post-translationally. The deduced amino-acid sequence shows significant homology to S-adenosylmethionine (SAM)-dependent methyltransferases. We hypothesize that the 27-kDa protein, PaMTH1, is involved in age-related methylation reactions protecting aging cultures against increasing oxidative stress. Received: 17 September / 1 December 1999     

Mitochondrial DNA of Chlamydomonas reinhardtii: the ND4 gene encoding a subunit of NADH dehydrogenase

Summary The ND4 gene encoding a subunit of respiratory NADH dehydrogenase has been identified on the linear 15.8 kb mitochondrial DNA of Chlamydomonas reinhardtii. The gene maps downstream of ND5. The 1,332 bp nucleotide sequence presented is the first complete reported ND4 sequence from a photoautotrophic organism. The deduced protein of 443 amino acid residues shows 34%, 29% and 27% homology to the protein sequences of Aspergillus amstelodami, Drosophila yakuba and mouse, respectively. ND4 is the fifth and last mitochondrial gene of the NADH dehydrogenase complex on the 15.8 kb mitochondrial genome of C. reinhardtii.     

Characterization of the nuclear gene encoding chloroplast ribosomal protein S13 from Arabidopsis thaliana

We have characterised a cDNA clone and a nuclear gene encoding the chloroplast 30 s ribosomal protein S13 from Arabidopsis thaliana. The identification is based on the high similarity of the predicted amino-acid sequence with eubacterial S13 protein sequences, and immunodetection of a 14.5-kDa chloroplast ribosomal polypeptide using antibodies raised against the polypeptide produced from part of the cDNA expressed in bacteria. The predicted amino-acid sequence contains an N-terminal extension which has several features characteristic of chloroplast transit peptides. Experiments suggest there is a single copy of this gene in A. thaliana and multiple copies in Brassica species. The origin of the mitochondrial S13 polypeptide in crucifers is also discussed.     

Analysis of the tandem repeat locus D4Z4 associated with facioscapulohumeral muscular dystropothhy

The sequence of the tandem repeat sequence (D4Z4) associatedwith facioscapulohumeral muscular dystrophy (FSHD) has beendetermined: each copy of the 3.3 kb repeat contains two homeoboxesand two previously described repetitive sequences, LSau anda GC-rich low copy repeat designated hhspm3. By Southern blotting,FISH and isolation of cDNA and genomic clones we show that thereare repeat sequences similar to D4Z4 at other locations in thehuman genome. Southern blot analysis of primate genomic DNAindicates that the copy number of D4Z4-like repeats has increasedmarkedly within the last 25 million years. Two cDNA clones wereisolated and found to contain stop codons and frameshifts withinthe homeodomains. An STS was produced to the cDNAs and analysisof a somatic cell hybrid panel suggests they map to chromosome14. No cDNA clones mapping to the chromosome 4q35 D4Z4 repeatshave been Identified, although the possiblilty that they encodea protein cannot be ruled out. Although D4Z4 may not encodea protein, there is an association between deletions withinthis locus and FSHD. The D4Z4 repeats contain LSau repeats andare adjacent to 68 bp Sau3A repeats. Both of these sequencesare associated with heterochromatic regions of DNA, regionsknown to be involved in the phenomenon of position effect variegation.We postulate that deletion of D4Z4 sequences could produce aposition effect.     

Evolutionary conservation of genomic sequences related to the GGP1 gene encoding a yeast GPI-anchored glycoprotein

Summary The GGP1 gene encodes the only GPI-anchored glycoprotein (gp115) that has been purified todate in the budding yeast Saccharomyces cerevisiae. It is a single-copy gene whose deduced amino-acid sequence shares no significant homology to any other known protein. In this paper we report a Southern hybridization analysis of genomic DNA from different eukaryotic organisms to identify homologues of the GGP1 gene. We have analyzed DNA prepared from a unicellular green alga (Chlamydomonas eugametos), from two distantly related yeast species (Candida cylindracea and Schizosaccharomyces pombe), and from the common bean Phasoleus vulgaris. The moderate stringency of the experimental conditions and the high specificity of the probes used indicate that a single-copy of GGP1-related sequences exists in all these eukaryotic organisms. The chromosomal localization of the GGP1 gene in S. cerevisiae has also been determined.     

Isolation of a cDNA probe for the human intestinal dipeptidylpeptidase IV and assignment of the gene locus DPP4 to chromosome 2

We report the nucleotide sequence and derived amino-acid sequence of a cDNA clone encoding the 3' end of human intestinal dipeptidylpeptidase IV (DPP-IV). This cDNA probe identifies a 4 kb mRNA in the human colon cancer cell line Caco-2. We demonstrate here an extensive homology between this human DPP-IV cDNA and the recently published rat liver DPP-IV cDNA. Using the human DPP-IV cDNA to probe genomic DNA from a panel of somatic cell hybrids we have assigned the gene encoding human DPP-IV to chromosome 2.     

Two amino-acid biosynthetic genes are encoded on the plastid genome of the red alga Porphyra umbilicalis

Summary To isolate the gene encoding the amino-acid biosynthetic enzyme acetolactate synthase (ALS) from the red alga Porphyra umbilicalis, PCR experiments were carried out using P. umbilicalis DNA as the template and degenerate oligonucleotides representing conserved regions of ALS amino-acid sequences. Interestingly, the PCR product (0.9 kb) hybridized exclusively to the plastid DNA of this red alga. DNA sequencing of two contiguous EcoRI plastid DNA clones revealed a 590 aminoacid open reading frame with 55 to 61% identity to cyanobacterial ALS sequences. A second gene (argB) encoding another amino-acid biosynthetic enzyme, Nacetylglutamate kinase, was identified upstream of, and on the opposite strand to the gene encoding ALS (ilvB). This is the first molecular characterization of a gene for an arginine biosynthetic enzyme from any plant. In addition, two tRNA genes, trnT(GGU) and trnY(GUA), were detected downstream from ilvB while four tRNA genes, trnfM(CAU), trnA(GGC), trnA(GGC), trnS(GCU) and trnD(GUC), were found downstream from argB. trnA(GGC) is not found in the chloroplast genomes of land plants.     

Characterization of DNA sequences constituting the terminal heterochromatin of the chicken Z chromosome

Two clones, pCZTH5-8 and pCZTH12-8, were isolated from a female chicken genomic library by screening with sequences obtained from genomic libraries which had been constructed from a terminal region of a single Z chromosome of chicken utilizing laser microbeam irradiation and PCR amplification. Fluorescencein situ hybridization to the mitotic Z chromosome and the lampbrush ZW bivalent of chicken demonstrated that both the cloned sequences are located in the heterochromatic region of the Z chromosome at the end opposite to the pairing region with the W chromosome. The sequences pCZTH5-8 and pCZTH12-8 are distributed widely on both the telomeric bow-like loops (TBL) and the region I (short loops region) of the Z lampbrush chromosome. These clones, pCZTH12-8 particularly notably, hybridized also to the TBLs of lampbrush bivalents 1–4 of chicken. Both sequence are transcribed in the lampbrush stage oocytes on the Z chromosome and on other macrobivalents. The subfragment of pCZTH5-8 which hybridizes to the TBLs and the insert of pCZTH12-8 contain regions that are closely similar in sequence. The pCZTH5-8 sequence has no internal repeats and may be part of the 24-kb macrosatellite repeating unit that is evident afterNhel digestion of the genomic DNA. A cloned 24-kb unit, pFN-1, does not show significant DNA curvature, but cytosines of its CpG dinucleotides may be highly methylatedin vivo. This contrasts with the repeat sequences of the W heterochromatin which not only have highly methylated CpG but are also strongly curved. The 24-kb unit is repeated about 830 times in the diploid genome of a female chicken, suggesting that nearly the entire terminal heterochromatin on the Z chromosome consists of this macrosatellite family. Sequences of the greater part of the pCZTH5-8 are restricted to the genusGallus but the sequence of one subregion which hybridizes to TBLs is present in the genomes of the order Galliformes.accepted for publication by M. Schmid     

Cloning and sequencing of cellulase cDNA from Aspergillus kawachii and its expression in Saccharomyces cerevisiae

The cDNA encoding the endo--1,4-glucanase (carboxymethylcellulase; CMCase-I) from Aspergillus kawachii IFO 4308 was cloned. Nucleotide-sequence analysis of the cloned cDNA insert showed a 717-bp open reading frame that encoded a protein of 239 amino-acid residues. The predicted amino-acid sequence of the mature protein had considerable homology with the protein sequence of the FI-CMCase of Aspergillus aculeatus. The cDNA was introduced into Saccharomyces cerevisiae. The expressed enzyme had carboxylmethylcellulase acitivity, identified by clear zones on a CMC-agar plate after Congo Red staining.